Abstract

The spindle pole body (SPB) represents the microtubule organizing center in the budding yeast Saccharomyces cerevisiae. It is a highly structured organelle embedded in the nuclear membrane, which is required to anchor microtubules on both sides of the nuclear envelope. The protein Spc72, a component of the SPB, is located at the cytoplasmic face of this organelle and serves as a receptor for the gamma-tubulin complex. In this paper we show that it is also a binding partner of the nuclear export receptor Xpo1/Crm1. Xpo1 binds its cargoes in a Ran-dependent fashion via a short leucine-rich nuclear export signal (NES). We show that binding of Spc72 to Xpo1 depends on Ran-GTP and a functional NES in Spc72. Mutations in this NES have severe consequences for mitotic spindle morphology in vivo. This is also the case for xpo1 mutants, which show a reduction in cytoplasmic microtubules. In addition, we find a subpopulation of Xpo1 localized at the SPB. Based on these data, we propose a functional link between Xpo1 and the SPB and discuss a role for this exportin in spindle biogenesis in budding yeast.

Xpo1 interacts with the SPB proteins Spc72 and Spc110 by the yeast two-hybrid method. (A) Summary of the results obtained in the yeast two-hybrid screen. The following proteins or protein fragments were identified as Gal4-activation domain fusions in the screen: Yap (residues 184 to 640), Nup42 (1 to 379), Nup49, Sgm1 (218 to 707), Sla2 (309 to 720), Spc72 (3 to 433), Ltv1 (55 to 463), and Hpa3. No interaction can be detected between Gal4-AD and BD-Xpo1 in the empty vector control. For simplicity, gene names rather than protein fragment length were used. Interactions were tested on SD-Trp−-Leu−-His− plates. (B) Xpo1 interacts with the N terminus of Spc110 but not with components of the γ-tubulin complex. Gal4-AD fusions of full-length Spc97, Spc98, and Tub4 show no interaction when tested against BD-Xpo1. Gal4-AD-Spc110 comprises amino acids 1 to 204 from the N terminus of this protein (Spc110204). For Gal4-AD fusions of Spc72, a 271-amino-acid (residues 1 to 271; Spc72271) and a 430-amino-acid (residues 3 to 433; Spc72433) fragment of the N terminus were tested. Interactions were tested on SD-Trp−-Leu−-His− plates.

Some fraction of Xpo1 localizes at the SPB in vivo. (A) Xpo1-GFP (strain GSY835) and Xpo1-1-GFP (KSY69 []) localize at distinct dots at the nuclear periphery (top). Xpo1-YFP and Spc29-CFP colocalize (Merge) at the SPB (KSY456) (bottom). Bar, 5 μm. (B) Xpo1-GFP is not lost from the SPB in an spc72Δ strain. Shown is a comparison of Xpo1-GFP localization in SPC72 (KSY460) and an spc72Δ mutant (KSY461). In the spc72Δ strain, two nuclei are present in one cell, indicative of a defect in nuclear migration (). Bar, 5 μm. (C) Perturbations in nuclear Gsp1-GTP concentration influence binding of Xpo1-YFP at the SPB. Plasmid-borne WT (Gsp1) and mutant forms of Gsp1 locked in either the GTP-bound (Gsp1-G21V) or nucleotide-free (Gsp1-T26N) state, respectively, were overexpressed from the GAL promoter in KSY456. A total of 100 random cells showing an Spc29-CFP signal were inspected for the presence or absence of Xpo1-YFP at the SPB, as described in the Materials and Methods section. Error bars (standard deviations) are shown. DIC, differential interference contrast optics.

Xpo1 binds a fragment of Spc72 in a Gsp1- and NES-dependent manner in vitro. (A) GST-NES as a control or GST-Spc72433 (GST fused to amino acids 3 to 433 of Spc72) (18 μg per reaction) was immobilized on GSH-Sepharose and incubated for 30 min at 4°C with 15 μg of recombinant Gsp1-GDP and Gsp1-GTP and 12 μg of Xpo1 as indicated. After binding, the supernatant (S) was precipitated with trichloroacetic acid and resuspended in sodium dodecyl sulfate sample buffer. Bound material was washed three times and subsequently eluted from the beads (B) with sodium dodecyl sulfate sample buffer. All samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie blue staining. (B) As in panel A, GST-Spc72433 was immobilized on GSH beads and incubated with Gsp1-GTP and Xpo1. In addition, synthetic peptides comprising either the WT PKI-NES (89 μg) or a mutated version thereof (NES*; 85 μg) were included in the binding reaction. Samples were further processed as described in panel A. M, molecular mass (in kilodaltons).

Spc72433 contains a leucine-rich NES-like amino acid motif at position 418 to 429 that mediates binding to Xpo1 in vitro and in vivo. (A) Schematic drawing of the full-length Spc72 sequence with known binding partners (black bars) and 12 putative NES-like motifs (black squares) indicated. Regions shown in dark gray indicate putative coiled-coil motifs. Spc72 fragments of indicated lengths were expressed as GST fusions in E. coli and used in the binding assays summarized in panel B. (B) Binding assay with Spc72 fragments. Binding assays were done exactly as described in the legend of Fig. , and only binding to the beads is shown. GST fusions of Spc72 fragments of indicated lengths (lanes 3 to 8; 18 μg each) were bound to GSH beads and incubated with Gsp1-GTP (15 μg) and Xpo1 (12 μg). Lane 8 contains an NES-mutated version of GST-Spc72433 (Spc72433*). As positive and negative controls for binding, GST-PKI-NES (18 μg; lane 1) and GST alone (18 μg; lane 2) are shown. (C) Yeast two-hybrid analysis of Spc72 fragments and full-length proteins. PJ69-4a cells were transformed with either pGBD or pGBD-Xpo1, respectively, and a WT (pGAD-Spc72433) or NES-mutated (pGAD-Spc72433*) version of Spc72433. pGAD-Spc72 and NES-mutated pGAD-Spc72* were also tested. Interactions were tested on SD-Trp−-Leu−-His− medium (SD −TLH). Plasmid-dependent growth was controlled on SD-Trp−-Leu− medium (SD −TL).

The leucine-rich NES-like sequence at position 418 to 429 of Spc72 mediates nuclear export in vivo. (A) Summary of the results obtained from a nuclear export reporter assay in vivo. The NES sequence from PKI (NLS-PKI-NES-GFP; pKW430) and the putative Spc72 NES LEKQINDLQIDK418-429 (NLS-SPC72-NES-GFP; pKS195) were expressed as GFP fusions in living yeast. In XPO1 cells (KWY120), due to the NES and NLS, the proteins shuttle between the nucleus and cytoplasm at 37°C and can be localized to both compartments (GFP, upper row). However, in the xpo1-1 temperature-sensitive mutant (KWY121), export activity is slowed down substantially at 37°C (GFP, lower panels), and therefore even in the presence of an intact NES, the reporter proteins accumulate in the nucleus. Bar, 5 μm. (B) Localization of GFP-tagged N and C termini of Spc72. The N-terminal 433 amino acids of Spc72 (NtermSPC72-GFP) were fused to GFP in either their WT (NES; pKS227) or NES-mutated (NES*; pKS228) version. The Spc72 C terminus comprising amino acids 396 to 622 (NLS-CtermSPC72-GFP) was fused to two moieties of GFP either in its WT (NES; pKS225) or NES-mutated (NES*; pKS226) version. In addition, the C-terminal fusion proteins contain an artificial NLS sequence to allow for nuclear accumulation. All fusion proteins were expressed in cells WT for XPO1 and SPC72 (KSY57) at 25°C. Bar, 5 μm. DIC, differential interference contrast optics.

Mutations in the NES sequence of Spc72 influence its binding to the SPB and affect spindle morphology in vivo. (A) Localization of WT (Spc72-GFP) and NES-mutated Spc72-GFP (Spc72*-GFP) in vivo. The upper row shows z-axis plane fluorescence images of a WT yeast cell in which SPC72 is genomically tagged with GFP (KSY457). The two lower rows show individual cells of a strain (KSY458) in which the NES418-429 of Spc72-GFP has been mutated as described in Materials and Methods. Exposure time was the same for all samples. For comparison, the position of the cell nucleus is indicated by DAPI staining. Bar, 5 μm. (B) Fluorescence intensity quantification of Spc72-GFP at the SPB. Haploid strains of SPC72 and XPO1 alleles were grown to mid-log phase and processed for fluorescence intensity quantification as described in Materials and Methods. The left panel compares the GFP fluorescence intensity at the SPB of a WT (Spc72-GFP; strain KSY457) and an NES-mutated (Spc72*-GFP; strain KSY458) version of Spc72-GFP. The right panel shows a similar experiment in which Spc72-GFP fluorescence was measured at SPBs of three different XPO1 alleles: WT (KSY582), xpo1-1 (KSY583), and xpo1-101 (KSY584). The mean intensity ± standard deviation of at least 30 SPBs is shown for each strain. (C) The formation of cytoplasmic MT (cyt. MT) is impaired in an spc72* mutant in vivo. For SPC72 WT (KSY462), spc72* mutant (spc72*; KSY463), another WT (KSY451), and spc72Δ strains (KSY452), a GFP-tagged copy of TUB1 was integrated at the URA locus. In each experiment, 200 cells in the G1 phase of the cell cycle (see example cells) were visually inspected for the presence or absence of cytoplasmic MTs as described previously (). Error bars (standard deviations) are shown. Size bar, 5 μm. (D) Formation of cytoplasmic MTs is reduced in XPO1 alleles in vivo. GFP tagging of TUB1 in XPO1 (KSY453), xpo1-1 (KSY454), and xpo1-101 (KSY455); temperature shifts; and cell classification were done as described in Materials and Methods. DIC, differential interference contrast optics.